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Experiments and theory about an elementary coding system based on RNA

Experiments and theory about an elementary coding system based on RNA Brookhaven Laboratory 01/13/2008. Jean Lehmann Center for Studies in Physics and Biology The Rockefeller University, New York. The genetic code. Lehmann 2006 Springer Verlag.

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Experiments and theory about an elementary coding system based on RNA

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  1. Experiments and theory about an elementary coding system based on RNA Brookhaven Laboratory 01/13/2008 Jean Lehmann Center for Studies in Physics and Biology The Rockefeller University, New York

  2. The genetic code Lehmann 2006 Springer Verlag

  3. The three chemical reactions required for translation:1) Activation of the amino acid (aa): aa + ATP  aa-AMP + ppiDG0 ≈ 02) Esterification of the tRNA: RNA + aa-AMP  aa-RNA + AMP3) Translation: peptide-RNA1 + aa-RNA2DG0 < 0 RNA1 + (peptide + 1)-RNA2 existing ribozymes

  4. Research goals The main goal of this research is to establish a minimal form of the translation process based on small RNA structures, without proteins. It is expected that a simplified geneticcode will be associated with this polymerization process. Theoretical challenge: Make a bridge between the laws of kinetics and thermodynamics, relevant to describe the events at the molecular level, and a theory of coding.

  5. Major Steps of the research • 1) Understand the structural requirements for an RNA • to load an amino acid without enzyme • 2) Once these RNAs will be isolated, establish • a translation system compatible with them

  6. Major Steps of the research • 1) Understand the structural requirements for an RNA • to load an amino acid without enzyme • 2) Once these RNAs will be isolated, establish • a translation system compatible with them

  7. Small RNAstem-loop http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect04.htm

  8. Folding of small random RNA sequences (nucleotides) Lehmann et al., 2004. J. theor. Biol. 227:381-395

  9. Self-aminoacylating ribozymes size: 29 nucleotides Illangasekare and Yarus 1999. RNA 5, 1482-1489

  10. Aminoacylation mechanism 3’ extension NaCl 100 mM MgCl2 80 mM CaCl2 40 mM 0ºC pH 7.0 k2nd (modern tRNA) ~ 25 nucleotides ~ 75 nucleotides

  11. Mass spectroscopy HPLC analysis of ribozyme activity

  12. 3’ extensions :GUUACG (squares)GUUUUACG (triangles)GUUUUUUACG (circles) Kinetics of aminoacylation Lehmann et al., 2007. RNA 13:1191-1197 Solution:

  13. Influence of the bases in the extension Lehmann et al., 2007. RNA 13:1191-1197

  14. (the smallest ribozyme) Lehmann et al. in prep.

  15. Extending the catalytic repertoire of self-aminoacylating ribozymes 1) Activation of the amino acid (aa): aa + ATP  aa-AMP + ppiDG0 ≈ 02) Esterification of the tRNA: RNA + aa-AMP  aa-RNA + AMP3) Translation: peptide-RNA1 + aa-RNA2DG0 < 0 RNA1 + (peptide + 1)-RNA2 existing ribozymes

  16. Extending the catalytic repertoire of self-aminoacylating ribozymes 1) Activation of the amino acid (aa): aa + ATP  aa-AMP + ppiDG0 ≈ 02) Esterification of the tRNA: RNA + aa-AMP  aa-RNA + AMP3) Translation: peptide-RNA1 + aa-RNA2DG0 < 0 RNA1 + (peptide + 1)-RNA2 wanted ribozyme

  17. Possible form of the wanted ribozyme Original ribozyme

  18. Major Steps of the research • 1) Understand the structural requirements for an RNA • to load an amino acid without enzyme • 2) Once these RNAs will be isolated, establish • a translation system compatible with them

  19. Major Steps of the research • 1) Understand the structural requirements for an RNA • to load an amino acid without enzyme • 2) Once these RNAs will be isolated, establish • a translation system compatible with them

  20. The genetic code Lehmann 2006 Springer Verlag

  21. A correlation in the genetic code: physico-chemical constraints at the level of translation codons Lehmann, 2000. J. theor. Biol 202:129-144

  22. Lightstone and Bruice, 1996J. Am. Chem. Soc.118, 2595 Influence of neighboring groups on the rate of a chemical reaction

  23. Analytical model for an elementary translation: Two amino acids, two anticodon-codon couples a priori configurations k+ k–(2) amino acid codon translation

  24. Two parameters fixed: a = 100 c = 0.01 coding phenomenon observed if b ~ 1

  25. Our analysis provides some answers to the origin of the correlation (and the code!) two codons, two amino acids: ~80% ~20% Parameters involved: b, c ~80% ~20% a ~ 100 b ~ 1 c ~ 0.01 d ~ 0.1 Parameters involved: a, b, d Lehmann, in prep.

  26. Our analysis provides some answers to the origin of the correlation (and the code!) two codons, two amino acids: ~80% ~20% Parameters involved: b, c ~80% ~20% a ~ 100 b ~ 1 c ~ 0.01 d ~ 0.1 Parameters involved: a, b, d Lehmann, in prep.

  27. Conclusions and outlook Part 1: We now better understand the structural features enabling small RNAs to covalently attach (activated) amino acids (reaction 2). The coupling between activation (reaction 1) and aminoacylation (reaction 2) still needs to be demonstrated. Part 2: A correlation shows that the crossing of the last chemical step leading to polymerization (reaction 3) is conditioned by physico-chemical constraints. The establishment of a translation system compatible with the ribozymes studied in Part 1 still needs to be demonstrated.

  28. Acknowledgements: Albert Libchaber Shixin Ye Axel Buguin Hanna Salman Carine Douarche Yusuke Maeda Funding: Swiss National Science foundation (Fellowships I & II ) The Rockefeller University (M.J & H. Kravis Fellowship)

  29. Biological Evolution Piece of information ( I) (DNA or RNA) Effect of the protein on the organism  Selection decoding process DP -code- Phenotype (protein) protein =DPcode ( I) Selection =f(protein) =f°DPcode ( I) If the code is not unique, there are as many possible selections on a given I as there are ways of decoding DP. Selection cannot operate at the same time on the information and on the code. The original code is therefore not the product of selection.

  30. Activation step as catalyzed by a Synthetase (amino acid = glycine) Arnez et al., 1999 J. Mol. Biol 286, 1449-1459

  31. Activation step as catalyzed by a Synthetase (amino acid = glycine) glycine ATP Arnez et al., 1999 J. Mol. Biol 286, 1449-1459

  32. Activation step as catalyzed by a Synthetase (amino acid = glycine) glycine-AMP ppi Arnez et al., 1999 J. Mol. Biol 286, 1449-1459

  33. Poly-U in the extension: effect of the length D, l: parameters a : length monomer lp : persistence length n : nb monomers Probability that the end of the extension lies on the binding site within a small interval of time t:

  34. Miller - prebiotic synthesis experiment

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